Recent explosion in genomic medicine studies has led to identification of an increasing number of genomic variations from patients with congenital defects. However, detailed analyses about how the variants affect developmental processes are scarce, resulting in a growing class of variants classified as variants of uncertain significance (VUS). Understanding functional consequences of VUS will help patients, their families, and their doctors to learn genetic underpinnings of their conditions, enable clinicians to provide personalized medical care and treatment, and expand our knowledge on biology and mechanistic operations of the molecules involved in disease processes. In this proposal, we plan to employ multidisciplinary approaches to investigate the YWHAZ variants associated with congenital syndromes. Our preliminary studies on one variant identified from a patient with RASopathy revealed that the variant activated the RAF-ERK pathway more efficiently than wild type YWHAZ in a vertebrate animal model, the African clawed frog Xenopus. The results show for the first time that YWHAZ variant may contribute to etiology of RASopathies. Several other YWHAZ variants also associate with human disorders, but functional significance of the variants has not been examined. Our proposed research will leverage the advantages of the Xenopus model, the power of biochemical and structural studies, and the strength of the mouse genetic system for detailed characterization of the YWHAZ variants. We will investigate whether and how YWHAZ variants have altered activities (aim 1), how sequence variations affect protein structure and interaction (aim 2), and how the variants induce pathology in the mouse models (aim 3). The novel combination of our investigation teams with expertise in distinct research disciplines promises generation of profound insight into disease- associated YWHAZ gene function and mechanisms.